This grant seeks continued support for our research on the mechanism of enzyme action. Specifically, we propose to investigate three enzymes which are involved in the metabolism of three-carbon units. Our work on glyoxalase I will focus on the identification of the active site base responsible for proton transfer using some of the principles of our recent finding of "mirror-image" catalysis by glyoxalase I. In addition, other studies are designed to probe the nature of the partitioning reaction of halomethylglyoxals by the enzyme. We will attempt to determine the contributions of product release from the enzyme and halogen orientation on the partitioning reaction. Our studies on lactate racemse will focus on two major issues. First, we will investigate the question of proton versus hydride transfer in the racemization step. We will accomplish this using many of the techniques that we have established in the glyoxalase I studies. Second, we will address the question of substrate activation in this system. There has been some speculation that lactate racemase may involve a covalent enzyme-substrate complex or a "charged" enzyme. Extreme difficulty in the purification and stabilization of this enzyme has prevented the resolution of this issue. We propose to develop a general method for the detection of a "charged" enzyme which we call anhydride detection by intermolecular oxygen scrambling. The technique relies on the implied radomization of carboxylate oxygens in an acyl exchange mechanism. Our technique will involve a novel utilization of 18O perturbations on 13C NMR resonances. This method will be extended to other enzymes where "charged" forms and anhydride intermediates have been proposed. Finally, we would like to initiate studies on elucidating the molecular mechanism of pyruvate-formate lyase, an enzyme which bears some similarity to the two discussed above in terms of ambiguity of electron flow. We propose studies designed to determine the direction of bond cleavage, the intermediates formed on the enzyme, and the function of enzyme activation by SAM, flavodoxin, and Fe(II)-protein complex.